Abstract
We describe detailed methods to measure thymidine (dThd) and deoxyuridine (dUrd) concentrations and thymidine phosphorylase (TP) activity in biological samples. These protocols allow the detection of TP dysfunction in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Since the identification of mutations in TYMP, the gene encoding TP, as the cause of MNGIE (Nishino et al. Science 283:689–692, 1999), the assessment of TP dysfunction has become the best screening method to rule out or confirm MNGIE in patients. TYMP sequencing, to find the causative mutations, is only needed when TP dysfunction is detected. dThd and dUrd are measured by resolving these compounds with high-performance liquid chromatography (HPLC) followed by the spectrophotometric monitoring of the eluate absorbance at 267 nm (HPLC-UV). TP activity can be measured by an endpoint determination of the thymine formed after 1 h incubation of the buffy coat homogenate in the presence of a large excess of its substrate dThd, either spectrophotometrically or by HPLC-UV.
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References
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Acknowledgment
Dr. Martí is supported by a grant from the Spanish Instituto de Salud Carlos III (PS09/01591). Dr. López is supported by grants from the Marie Curie International Reintegration Grant Programme (COQMITMEL-266691) of the Seventh European Community Framework Programme, from Ministerio de Ciencia e Innovación, Spain (SAF2009-08315) and from the Consejería de Economía, Innovación y Ciencia, Junta de Andalucía (P10-CTS-6133). Dr. Hirano is supported by NIH grants R01 HD056103 (cofunded by NICHD and the NIH Office of Dietary Supplements), R01 HD057543, RC1 NS070232; MDA grant 115567; and the Marriott Mitochondrial Disorder Clinical Research Fund.
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Martí, R., López, L.C., Hirano, M. (2012). Assessment of Thymidine Phosphorylase Function: Measurement of Plasma Thymidine (and Deoxyuridine) and Thymidine Phosphorylase Activity. In: Wong, Ph.D., LJ. (eds) Mitochondrial Disorders. Methods in Molecular Biology, vol 837. Humana Press. https://doi.org/10.1007/978-1-61779-504-6_8
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DOI: https://doi.org/10.1007/978-1-61779-504-6_8
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